The polymerase chain reaction (PCR) is a very powerful method for the specific amplification of DNA or RNA stretches. The methodology is described in European Patent Applications, Pub. Nos. 201.184, 200.362 and 258.017. One application of this technology is in DNA probe technology to bring up DNA present in low copy numbers to a detectable level. Numerous diagnostic and scientific applications of this method have been described by H. A. Erlich (Ed.) in PCR Technology-Principles and Applications for DNA Amplification, Stockton Press, USA, 1989 and by M. A. Inis (Ed.) in PCR Protocols, Academic Press, San Diego, USA, 1990.
A desirable goal would be the direct detection of the amplified DNA without time-consuming separations or transfer steps by a so-called homogeneous assay format. At the same time the aim is also to replace radioactive labels still mainly utilized in DNA diagnostics by nonradioactive reporter systems thereby extending the applications of this technology. Such a homogeneous detection system applying intercalating chemiluminescent acridinium esters has been reported by Arnold et al. in Clinical Chemistry 35, 1588 (1989). Further variations of the homogeneous DNA detection assays are described in a review by B. S. Reckmann in Nachr. Chem. Tech. Lab. 37, 692-702 (1989).
The use of bathophenanthroline-Ru II complexes as nonradioactive label molecules which can be measured with high sensitivity by time-resolved fluorometry has been described by W. Bannwarth et al. in Helv. Chim. Acta 71, 2085-2099 (1988). These complexes can be part of an interactive pair of label molecules allowing energy transfers from suitable energy donor molecules to the Ru complex. Because the efficiency of the energy transfer is highly dependent on the distance between donor and acceptor molecules, such energy transfer systems are useful in studying molecule interactions.
A suitable class of donor molecules for use with Ru complexes is the lumazine chromophore group of molecules. The possible applicability of this donor/acceptor complex in the detection, inter alia, of DNA molecules in a homogeneous assay is described in European Patent Application, Publ. No. 439 036, and in Helvetica Chimica Acta 74, 1991-1999 (1991) and 74, 2000-2008 (1991) by W. Bannwarth and F. M uller. Using such a combination, energy transfers were detected within oligodeoxynucleotides labeled at the 5'-end with a Ru bathophenanthroline complex and possessing lumazine chromophores at different distances from the Ru complex within the oligonucleotide. It also was demonstrated that this pair of interactive labels is useful in detecting a target DNA sequence in a hybridization process wherein one probe sequence is equipped with the donor and the other with the acceptor.
An alternative approach using a terbium complex as an energy acceptor and salicylate as an energy donor in a homogeneous DNA detection system was described by A. Oser and G. Valet in Angewandte Chemie 102, 1197-1200 (1990).
Known processes for the detection of oligonucleotides in a homogeneous test format employing energy transfers for the subsequent detection typically use at least two labeled oligonucleotides that hybridize specifically, side by side, to the complementary DNA sequence thereby positioning the two labels next to each other.